A great deal of research has been directed to the development of accurate techniques for determining the presence of organic materials such as drugs, drug metabolites, contaminants, pollutants, and the like in substances of interest such as food, soil, and bodily fluids. For example, pathological or other conditions in human beings and animals are often detected by performing immunoassays on samples of bodily fluids such as urine or blood serum. Immunoassays are based on the capacity of a first compound, known as a ligand, to recognize or bind a second compound, known as a receptor, having a specific spatial and/or polar organization. Typically, immunoassays are used for the detection of antibodies, antigens, or haptens in bodily fluids.
Antigens are foreign substances which, when introduced into a higher animal, bring about the formation of antibodies which react with the antigens to initiate protection against infection or disease. A single antigen may contain multiple antigenic determinants, also known as active sites, which are regions of the antigen molecule that specifically elicit the production of antibody to which the antigenic determinant binds.
Antigens are to be contrasted with haptens, which are relatively small molecules that cannot alone elicit the production of antibodies. A hapten can act as an antigenic determinant and elicit antibody synthesis only when covalently attached to a larger carrier molecule. However, when detached from its carrier, the hapten will retain its ability to bind strongly to the antibody, albeit via what is believed to be a single active site.
The presence of an antigen, hapten, or antibody in a sample of bodily fluid typically can be confirmed or determined by contacting the corresponding antibody or the corresponding antigen with the sample. The presence or absence of the antigen, hapten, or antibody in the sample is usually established by detecting the occurrence or nonoccurrence of a reaction between the specific ligand/receptor pair. For example, the reaction between an antibody and an antigen usually manifests itself by insolubility or agglutination.
Because most ligand/receptor pairs are detected only with difficulty, it is frequently necessary to use certain inert carrier moieties to facilitate their detection. For example, in certain latex agglutination techniques an antibody and/or an antigen is covalently bound to discrete latex particles having diameters on the order of about 0.01 to about 100 micrometers. These particles are cross-linked or otherwise aggregated by the complementary antigen or antibody by way of the multiple binding sites found on both moieties. The agglutination of such particles into relatively large aggregates or clumps is then observed.
A slightly different situation is presented in detecting a hapten by latex agglutination. Since haptens possess but a single active site, haptens and their complementary antibodies do not "cross-link" or form long aggregates. Thus, it is necessary in latex agglutination techniques for the detection of haptens that the hapten be bound to the latex particles. The hapten present in a sample typically is then detected by its capacity to inhibit the agglutination of antibody-bearing particles and hapten-bearing particles. In such systems, a negative test for hapten is manifested by agglutination of antibody- and hapten-bearing particles and a positive test is manifested by the absence of such agglutination.
One serious problem with hapten detection by such techniques is that hapten-bearing particles are somewhat unstable and will often agglutinate with one another to form multiple-particle aggregates. Thus, it is known in the art to stabilize mixtures containing hapten-bearing latex particles with stabilizing factors, such as bovine serum albumin. However, these stabilizing factors are sometimes diluted by urine or serum during the course of a latex immunoassay, leading to agglutination of the hapten-bearing particles, even in the absence of antibody-bearing particles. Agglutination in this manner makes it difficult or impossible to effectively determine the presence of hapten in many types of samples.
The different types of latex agglutination techniques presently known in the art may be categorized into three basic classes based upon the particular method employed for detecting ligand/particle aggregates. The techniques of the first class involve centrifugation. For example, U.S. Pat. No. 4,738,932 in the name of Yabusaki discloses a centrifugation technique which involves rotating an agglutination slide on a serological rotator and then using a magnifier to examine slide wells for agglutination.
The second class comprises techniques which detect aggregates by particle counting. For example, Masson, et al., Methods in Enzymology, 1981, 74, 106-139, disclose an immunoassay technique in which a complicated device which uses forward light scattering is employed to count unaggregated particles. Thus, both the centrifugation and particle counting techniques have the disadvantages of complicated, time-consuming procedures and expensive, highly specialized devices.
The techniques of the third class are those in which agglutination is detected visually. However, since the average human eye can only detect particles down to about 40 micrometers in diameter, most visual agglutination tests must produce relatively large aggregates which typically require an undesirably long time to form. U.S. Pat. No. 4,459,361, in the name of Gefter, discloses a somewhat improved type of visual technique which involves visual detection of unaggregated particles rather than aggregated particles. In certain techniques according to Gefter, both the ligand and the receptor are separately immobilized on latex particles and then mixed with a sample of bodily fluid suspected to contain the ligand. In other techniques, the sample is incubated with the receptor and with latex particles bearing the ligand. According to Gefter, the ligand in the sample competes with the ligand-bearing particles for the receptor sites. To the extent that the ligand contained in the sample binds to the free receptor or receptor-bearing particles, the ligand-bearing particles fail to aggregate. Thus, when such a mixture is exposed to a filter having a controlled pore size there is a substantial increase in the amount of unaggregated, ligand-bearing particles which pass through the filter. It is the presence of these unaggregated particles which is then detected.
According to Gefter, the amount of unaggregated particles which pass through the filter is proportional to the amount of ligand in the sample. Further, Gefter states that the number of unaggregated particles is sufficient to be visible to the naked eye, and that this visibility can be enhanced by the selection of the size, color, optical density, or fluorescence of the particles.
However, the techniques disclosed by Gefter have a number of serious shortcomings. For example, certain techniques require that both ligand- and receptor-bearing particles be prepared, thus adding considerable time and expense. While Gefter asserts that the unaggregated, ligand-bearing particles can be detected with the naked eye, the provided examples do not detect such particles visually, but rather with sophisticated spectrophotometric devices. Moreover, the teachings of Gefter do not appear applicable where the ligand is a hapten. To the extent that hapten contained in a sample binds to antibody, hapten-bearing particles would be expected to aggregate and effect a decrease, rather than an increase, in the amount of unaggregated, hapten-bearing particles which pass through the filter.
Accordingly there still exists a need for relatively simple, inexpensive techniques for the accurate detection of biologically important chemical compounds such as drugs, drug metabolites, and other haptens which may be present in bodily fluids.